Lee Chi-Hsin, Liu Chia-I, Leu Sy-Jye, Lee Yu-Ching, Chiang Jen-Ron, Chiang Liao-Chun, Mao Yan-Chiao, Tsai Bor-Yu, Hung Ching-Sheng, Chen Chi-Ching, Yang Yi-Yuan
School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan.
Graduate Program in Medical Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan.
J Venom Anim Toxins Incl Trop Dis. 2020 Nov 20;26:e20200056. doi: 10.1590/1678-9199-JVATITD-2020-0056.
The venom of bamboo vipers ( - TS), commonly found in Taiwan, contains deadly hemotoxins that cause severe envenomation. Equine-derived antivenom is a specific treatment against snakebites, but its production costs are high and there are some inevitable side effects. The aim of the present work is to help in the development of an affordable and more endurable therapeutic strategy for snakebites.
venom proteins were inactivated by glutaraldehyde in order to immunize hens for polyclonal immunoglobulin (IgY) antibodies production. After IgY binding assays, two antibody libraries were constructed expressing single-chain variable fragment (scFv) antibodies joined by the short or long linker for use in phage display antibody technology. Four rounds of biopanning were carried out. The selected scFv antibodies were then further tested for their binding activities and neutralization assays to TS proteins.
Purified IgY from egg yolk showed the specific binding ability to TS proteins. The dimensions of these two libraries contain 2.4 × 10 and 6.8 × 10 antibody clones, respectively. An increase in the titers of eluted phage indicated anti-TS clones remarkably enriched after 2 panning. The analysis based on the nucleotide sequences of selected scFv clones indicated that seven groups of short linkers and four groups of long linkers were identified. The recombinant scFvs showed significant reactivity to TS venom proteins and a cross-reaction to venom proteins. In studies, the data demonstrated that anti-TS IgY provided 100% protective effects while combined scFvs augmented partial survival time of mice injected with a lethal amount of TS proteins.
Chickens were excellent hosts for the production of neutralization antibodies at low cost. Phage display technology is available for generation of monoclonal antibodies against snake venom proteins. These antibodies could be applied in the development of diagnostic kits or as an alternative for snakebite envenomation treatment in the near future.
台湾常见的竹叶青蛇毒液（-TS）含有致命的血液毒素，可导致严重的中毒。马源抗蛇毒血清是治疗蛇咬伤的特效药物，但其生产成本高昂，且存在一些不可避免的副作用。本研究旨在帮助开发一种经济实惠且更持久的蛇咬伤治疗策略。
用戊二醛使毒液蛋白失活，以免疫母鸡产生多克隆免疫球蛋白（IgY）抗体。在进行IgY结合试验后，构建了两个抗体文库，分别表达通过短连接子或长连接子连接的单链可变片段（scFv）抗体，用于噬菌体展示抗体技术。进行了四轮生物淘选。然后进一步测试所选scFv抗体与TS蛋白的结合活性和中和试验。
从蛋黄中纯化的IgY显示出与TS蛋白的特异性结合能力。这两个文库的规模分别包含2.4×10和6.8×10个抗体克隆。洗脱噬菌体滴度的增加表明，经过两轮淘选后，抗TS克隆显著富集。基于所选scFv克隆的核苷酸序列分析表明，鉴定出七组短连接子和四组长连接子。重组scFv对TS毒液蛋白显示出显著的反应性，并与毒液蛋白发生交叉反应。在研究中，数据表明抗TS IgY提供了100%的保护作用，而联合scFv延长了注射致死量TS蛋白的小鼠的部分存活时间。
鸡是低成本生产中和抗体的优良宿主。噬菌体展示技术可用于产生针对蛇毒蛋白的单克隆抗体。这些抗体可在不久的将来应用于诊断试剂盒的开发或作为蛇咬伤中毒治疗的替代品。
